Hematopoietic Regulatory Factor EDAG Promotes Erythrocyte Maturation And Survival By Regulating Hsp70 Localization

First of all,we focus on the hematopoietic transcriptional regulator EDAG,which involved in the regulation of hematopoietic cell proliferation,apoptosis and differentiation.In the present study,we confirmed the interaction between EDAG and Hsp70 by immunoprecipitation,and mapped the interaction domain between them.It was further confirmed that EDAG/Hsp70/GATA1 could form endogenous complexes,which showed dynamic changes during erythroid differentiation.Forced expression of EDAG increased the nuclear localization of Hsp70 in EPO-induced CD34~+cells during erythroid differentiation and blocked the cytoplasmic translocation of Hsp70with EPO deprivation,and increased the expression level of GATA1.We also found that EDAG significantly inhibited the activity of Caspase-3 and inhibited the apoptosis of CD34~+cells after EPO removal in a Hsp70-dependent manner.Knockdown of EDAG enhanced cell apoptosis induced by EPO deprivation,which can be inhibited by Caspase-3 inhibitors.These results suggest that overexpression of EDAG can promote the localization of Hsp70 in umbilical cord blood CD34~+nucleus,inhibit the activity of Caspase-3,protect GATA1 from Caspase-3 cleavage,and promote erythroid cell survival.In order to reveal the role of EDAG/Hsp70/GATA1complex in erythroid related diseases,we investigated the effect of EDAG on erythroid progenitor cells in patients with myelodysplastic syndromes(MDS).We found that EDAG was down-regulated in bone marrow erythroid progenitor cells in patients with MDS.Overexpression of EDAG significantly promoted the accumulation of Hsp70 in the bone marrow CD34~+nucleus of MDS patients and upregulated the protein level of GATA1.In addition,overexpression of EDAG can increase the erythroid differentiation of bone marrow erythroid progenitor cells in MDS patients and resist cell apoptosis.These results suggest that EDAG can promote Hsp70 into the nucleus,upregulate GATA1 expression,and involved in the regulation of differentiation and apoptosis of bone marrow erythroid progenitor cells in MDS patients,providing a new potential strategy for the clinical treatment of MDS disease.In order to further study the function of EDAG,we used the zinc finger enzyme technology to establish the EDAG knockout mouse model.We found that EDAG knockout mice developed normally under steady state.Here we report that under lipopolysaccharide(LPS)stimulation condition,the survival rate of EDAG knockout mice was significantly lower after LPS stimulation,suggesting that EDAG knockout led to increased susceptible to LPS-induced endotoxin shock.We also found that EDAG knockout mice produced elevated levels of G-CSF and chemokines than wild-type mice after LPS injection.The proportion of peripheral blood neutrophils from EDAG knockout mice was also higher than that in wild type group.The severity of the damage of multiple organs induced by LPS treatment in EDAG knockout mice was much higher than wild type.Overall,our results reveal that EDAG regulates functions of hematopoietic stem cells and immune cells during bacterial infection.In addition,we explored the mechanism of CK8 on negative regulation of TLR/NF-?B signaling.Keratin 8(CK8)belongs to the type II keratin family and is the major compontent of the intermediate filaments of simple or single-layered epithelia.Here we report that down-regulation of CK8 in mice enhanced TLR-mediated responses,rendering mice more susceptible to LPS-induced endotoxin shock with reduced survival,elevated levels of inflammation cytokines and more severe tissue damage.We found that CK8 suppressed TLR-induced nuclear factor(NF)-?B activation and interacted with the adaptor tumor necrosis factor(TNF)receptor-associated factor 6(TRAF6)to prevent its polyubiquitination.Our findings demonstrate a novel role of CK8 in negative regulation of TLR/NF-?B signaling and highlight a previously unidentified nonclassical function for CK8 in limiting inflammatory responses.